This invention relates to the effect of amlodipine and atorvastatin, alone and in combination, on cholesterol crystal formation and the release of nitric oxide (NO) from endothelial cells.
Coronary artery disease (CAD) is the leading cause of mortality in the developed world, and is associated with substantial morbidity as well. Typically, the patient with CAD has several concomitant conditions, including hypertension, diabetes, and dyslipidemia, increasing overall risk for poor outcomes and complicating treatment. A therapeutic goal for the treatment of CAD is the development of drugs that can simultaneously target multiple underlying disease processes that contribute to atherosclerosis, thereby altering the course of the disease. Therefore, CAD therapy may have increased positive outcomes if the use of an antihypertensive agent and HMG-CoA reductase inhibitor was combined in a single delivery system.
Free cholesterol is an important structural component of the cell plasma membrane that modulates packing of phospholipid molecules, thus regulating lipid bilayer dynamics and structure. The cholesterol molecule is oriented in the membrane such that the long-axis lies parallel to the phospholipid acyl chains, increasing order in the upper acyl chain region of the membrane while decreasing packing constraints at the terminal methyl groups. During atherogenesis, however, increasing levels of cellular cholesterol lead to its abnormal deposition in the vessel wall and the formation of cholesterol crystals.
In animal models of atherosclerosis, it has been demonstrated that the cholesterol content of membranes associated with vascular smooth muscle and macrophage foam cells becomes elevated, resulting in the formation of discrete domains. These highly organized cholesterol structures, characterized by a unit cell periodicity of 34.0 xc3x85, appear to serve as nucleating sites for the formation of extracellular crystals. These domains have been previously described in model membrane systems. A recent study from our laboratory showed that cultured mouse peritoneal macrophage foam cells produced free cholesterol crystals that extend from intracellular membrane sites with various morphologies that include plates, needles and helices. With the use of x-ray diffraction approaches, the early stages of crystal formation could be identified in isolated membranes from these cells. Preventing crystal formation is an important goal as cholesterol in this state is practically inert and does not respond well to pharmacologic interventions that promote lesion regression.
In addition, the normal production of NO by the endothelium is critical for maintaining vascular function. During atherosclerosis, however, endothelial dysfunction effects a significant reduction in NO production, resulting in: 1) increased monocyte and LDL infiltration, 2) loss of smooth muscle cell function and abnormal proliferation, 3) increased oxidative stress, and 4) increased platelet aggregation. Pharmacologic interventions that restore endothelial function and NO metabolism have demonstrated benefit in the treatment of various cardiovascular disorders, including coronary artery disease.
However, no pharmaceutical composition currently exists that treats both hypertension and hyperlipidemia. Such a pharmaceutical composition would have several benefits. For example, the multiple risk factors for arterial and related heart disease that are often present in an individual patient could be targeted simultaneously. Additionally, the ease of taking one combined dosage could significantly enhance patient compliance with therapeutic regimens.
Therefore, it is an object of this invention to provide a combination therapy that will treat the multiple pathological processes involved in arterial and related heart disease. These include, but are not limited to, hypertension and hyperlipidemia. It is also an object of this invention to develop useful and convenient dosage levels and forms of such a combination therapeutic. Preferably, this pharmaceutical composition would have synergistic effects on these hallmarks of arterial and related heart disease, such that the individual effects of the components of this composition would be enhanced by their combination.
Thus, this invention encompasses a therapeutic goal for the treatment of CAD that entails the development of drugs that can simultaneously target multiple underlying disease processes that contribute to atherosclerosis, thereby altering the course of the disease. Therefore, using this invention, CAD therapy may have increased positive outcomes if the use of an antihypertensive agent and HMG-CoA reductase inhibitor was combined in a single delivery system.
Lipophilic agents that influence the organization of lipids may interfere with the early formation of cholesterol domains within the membrane, thereby disrupting crystal formation. The charged dihydropyridine amlodipine (AML) was used for this study as it has strong affinity for phospholipid constituents of atherosclerotic-like membranes, as compared to other calcium channel blockers (CCBs). This property of the drug is attributed to a distinct membrane location that facilitates strong electrostatic binding to the phospholipid phosphate groups, as indicated by x-ray diffraction, differential scanning calorimetry and nuclear magnetic resonance analyses. These distinct membrane interactions may underlie the ability of AML to reverse cholesterol-induced increases in the width of atherosclerotic membranes obtained from vascular smooth muscle cells and to interfere with oxidative modification of lipid constituents.
The various membrane biophysical effects of AML may contribute to its observed benefit in coronary artery disease, as demonstrated in the Prospective Randomized Evaluation of the Vascular Effects of Norvasc Trial (PREVENT). The results of PREVENT showed a reduction in cardiovascular morbidity associated with AML therapy, including a 42% reduction in the need for revascularization and 33% reduction in documented angina, as compared to placebo. AML therapy was also associated with significant slowing in the progression of carotid atherosclerosis, as measured with B-mode ultrasonographic assessments. As such clinical benefit has not been observed with other CCB analogs in similarly designed trials, it has been proposed that AML may have distinct antiatherosclerotic activity.
Hypolipidemic therapy has also been demonstrated to be very useful in reducing morbidity and mortality associated with CAD. Among HMG-CoA reductase inhibitors, atorvastatin calcium (AT) has been shown to be very effective as hypolipdemic therapy. In a recent clinical study involving patients with stable and advanced CAD, aggressive lipid-lowering therapy with AT significantly delayed the time to a first ischemic event and reduced, by 36%, the overall incidence of cardiovascular events, as compared to angioplasty with usual medical care.
Small angle x-ray diffraction approaches were utilized to examine the separate and combined effects of AML and AT on the formation of cholesterol monohydrate structures (d-space of 34.0 xc3x85) within the membrane bilayer. This question was investigated in membranes containing cholesterol at levels that reproduce atherosclerotic-like conditions. The results of this study demonstrated that these compounds interfered with the aggregation of cholesterol into separate intrabilayer domains in an unexpected and highly synergistic fashion. This observed effect could not be reproduced by the combination of these compounds with other related drugs. By disrupting the development of cholesterol crystals, this drug combination may facilitate the removal of excess sterol from the vessel wall by plasma high-density lipoproteins (HDL).
In addition, NO production from endothelial cells in rabbit aorta was systematically measured in the absence and presence of amlodipine, atorvastatin, and the combination of the two compounds. The results of these experiments demonstrated a dramatic synergistic effect of these compounds on NO stimulation. These findings indicate that these agents may effectively stabilize, in a synergistic fashion, NO in the cell by interfering with oxidative destruction.
Other objects, features, and advantages of the present invention will be apparent from the following Detailed Description of the Preferred Embodiments taken in conjunction with the accompanying drawings in which: